Practically every physiological role ascribed to the platelet requires calcium (Ca). There are some indications that platelet calcium is involved in the hemostatic reactions of release and aggregation, yet knowledge about calcium compartmentalization and the source of calcium in platelets is scant. A platelet membrane fraction has the ability to accumulate calcium in an ATP-dependent process. This activity may represent the in vivo process by which intact platelets regulate the cytoplasmic concentration of Ca and/or sequester Ca in an intracellular pool. Because the platelet's small size is a limiting factor in the in vivo localization of Ca transport or storage sites, the proposed study will utilize the isolated calcium transporting membranes to study Ca compartmentalization and flux. The research effort will be directed toward elucidating the direction of energy-dependent Ca movement and localizing the Ca transporting membranes in the intact platelet. The direction of energy-dependent Ca transport (efflux or influx) can be derived from knowledge about the orientation of the membrane vesicles. The proportion of right-side-out to inside-out membranes will be estimated by measuring the amount of sialidase-sensitive sialic acid relative to total sialic acid content of the membranes. Selective labelling of the plasma membrane of intact platelets prior to homogenization will provide a means of quantitating the plasma membrane content of subsequently prepared membrane fractions. Autoradiography of calcium C14-oxalate loaded vesicles will provide an estimate of the proportion of vesicles active in calcium uptake. The isolated Ca transport system will be used as an in vitro model to evaluate the effects of various physiological and pharmacological agents on calcium binding, transport and storage.